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CD28 Bioassay

Measure Potency and Stability of Biologics Designed to Activate CD28

  • 96- and 384-well plate compatible
  • Prequalified according to ICH guidelines
  • No primary cell culture required
  • Available in convenient thaw-and-use format

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$ 700.00
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CD28 Bioassay
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Simple Bioassay, Consistent Results

The CD28 Bioassay is a bioluminescent cell-based assay used to measure the potency and stability of ligands or antibodies that activate CD28. CD28 binds to the B7 family members CD80 and CD86 (collectively referred to as B7 for this bioassay) on antigen-presenting cells (APCs). Co-stimulation of T cells by CD28 activation initiates signaling cascades that result in AP-1 and NFκB transcription factor activation and nuclear translocation. These pathways significantly enhance T cell cytokine production—specifically, interleukin-2 (IL-2)—which promotes T cell proliferation, differentiation and survival.

Antibodies that agonize CD28 have the potential to enhance immune responses against cancer and chronic infection. The CD28 Bioassay reflects the mechanism of action (MOA) of biologics designed to activate CD28.

CD28 Bioassay Components

The thaw-and-use format of the CD28 Bioassay Core Kit (Cat.# JA6701, JA6705) offers convenience and minimizes variability. The assay consists of the genetically engineered cell lines:

CD28 (TCR/CD3) Effector Cells: Jurkat T cells endogenously expressing TCR, CD3 and CD28 receptors and engineered with a luciferase reporter driven by a CD28 pathway-dependent promoter.

Based on the antibody properties to be tested, the CD28 Bioassay can be conducted with either aAPC/CHO-K1 Cells (Cat.# JA9441, JA9445) or FcγRIIb aAPC/CHO-K1 Cells (Cat.# JA9331, JA9335) to determine if the agonist antibodies can activate CD28 in an FcγR-dependent manner. These cells must be purchased separately.

aAPC/CHO-K1 Cells (Cat.# JA9441, JA9445): CHO-K1 cells engineered to express a cell-surface protein designed to activate the TCR complex in an antigen-independent manner. Intended for use with FcγRIIb-independent antibodies or other biologics that do not require crosslinking for optimal activity.

FcγRIIb aAPC/CHO-K1 Cells (Cat.# JA9331, JA9335): CHO-K1 cells engineered to express a proprietary TCR-activating protein as well as human FcγRIIb. FcγRIIb serves as a crosslinking receptor that binds to antibody Fc domains and clusters agonist antibodies for maximal agonist activity.

The CD28 Effector Cells and FcγRIIb CHO-K1 Cells are also available in Cell Propagation Model format (CPM; Cat.# JA1072) as cryopreserved cells that can be thawed, propagated and banked for long-term use.

Workflow

Standard bioassay workflow with cultured cells
Improved bioassay workflow with thaw-and-use cells

Standardized Reagents Improve Results

  • Thaw-and-use format provides significant time and labor savings over traditional methods while reducing variability.
  • Kit includes all of the required reagents in standardized formats.
  • Easily amenable to high-throughput workflows.

Assay Principle

The CD28 Bioassay measures the potency and stability of ligands or agonist antibodies and other biologics that bind and activate CD28.

Representation of the CD28 Bioassay.

Representation of the CD28 Bioassay. The CD28 Bioassay for FcγRIIb-dependent agonist antibodies consists of two cell lines, CD28 Effector Cells and FcγRIIb aAPC/CHO-K1 Cells. In the absence of agonist antibody, CD28 is not activated and the luminescence signal is low. In the presence of FcγRIIb aAPC/CHO-K1 Cells, anti-CD28 antibody can be crosslinked, thereby enhancing induction of CD28 pathway-activating luminescence in a dose-dependent manner by addition of Bio-Glo™ Reagent and quantitation with a luminometer. CD28 Effector Cells can also be used with antibodies that are FcγRIIb-independent by substituting aAPC/CHO-K1 Cells.

Assay Performance Data

Accuracy and precision are critical to bioassays and the development of biologics. The CD28 Bioassay is both accurate and precise. Accuracy is a measure of how close results are to a target or reference value. Precision is a measure of repeatability. This assay was developed and prequalified according to ICH guidelines for accuracy and precision giving it the performance characteristics required for many applications of a bioassay used for antibody drug discovery research and development.

Accuracy: Measure of how close results are to a reference or "target" value.

IntraLab Precision: Measure of repeatability from multiple assays of the same homogeneous sample under the same operating conditions over a short time (same analyst; same lab; same day).

Intermediate Precision: Measure of repeatability of multiple assays of the same homogeneous sample with different analysts or equipment or on different days.

Linearity: Ability of the assay to provide measured values that are proportional to the concentration of the sample.

32454752-data-target-combined

The CD28 Bioassay Shows Precision, Accuracy and Linearity.

Parameter Results
Accuracy % Expected Relative Potency % Recovery
50 43.6
70 67.2
100 93.5
140 139.9
200 205.5
Repeatability (% CV) 100% (Reference) 4.9
Intermediate Precision (% CV)   6.3
Linearity (r2)   0.998
Linearity (y = mx + b)   y = 1.075x – 10.48
A 50–200% theoretical potency series of Control Ab, Anti-CD28, was analyzed in triplicate in three independent experiments performed on three days by two analysts using the CD28 Bioassay for FcγRIIb-dependent antibodies. Bio-Glo™ Luciferase Reagent was added, and luminescence was quantified using the GloMax® Discover System. Data were analyzed, and relative potencies were calculated after parallelism determination using JMP® software. Data were generated using thaw-and-use cells.

 

The CD28 Bioassay reflects the mechanism of action (MOA) and show specificity of biologics designed to activate CD28.
CD28 Bioassay Mechanism of Action

CD28 Effector Cells were incubated with FcγRIIb aAPC/CHO-K1 Cells in the presence of serial titrations of agonist antibodies as indicated. After a 5-hour induction, Bio-Glo™ Reagent was added and luminescence quantified using the GloMax® Discover System. Data were generated using thaw-and-use cells.

CD28 Bioassay Mechanism of Action

FcγRIIb aAPC/CHO-K1 Cells were added to a 96-well assay plate 18 hours prior to the assay. On the day of the assay, a titration of Control Ab, Anti-CD28 (Cat.# K1231), and CD28 Effector Cells was added. After a 5-hour induction at 37°C, Bio-Glo™ Reagent was added and luminescence measured using the GloMax® Discover System.

  • CD28 Bioassay, Core Kit, is available in 1X and 5X sizes.
  • FcγRIIb aAPC/CHO-K1 Cells (Cat.# JA9331, JA9335) are available separately.
  • aAPC/CHO-K1 Cells (Cat.# JA9441, JA9445) are available separately.
  • Control Ab, Anti-CD28 (Cat.# K1231), is available separately.

Specifications

You are viewing: JA6701 Change Configuration

What's in the box?

Item Part # Size

TCR/CD3 Effector Cells (IL-2)

J129A 1 × 0.8ml

Fetal Bovine Serum

J121A 1 × 4ml

RPMI 1640 Medium

G708A 1 × 36ml

Bio-Glo™ Luciferase Assay Buffer

G719A 1 × 10ml

Bio-Glo™ Luciferase Assay Substrate

G720A 1 × 1 vial

SDS

Choose language:

Download SDSPDF (313 KB) – English (United States)

Certificate of Analysis

Search by lot number

Use Restrictions

Not For Medical Diagnostic Use.

Storage Conditions

Please see the Protocol for detailed storage conditions.

Patents and Disclaimers

NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE STATEMENT. If the recipient is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the recipient with a full refund.

This product may not be further sold or transferred by the recipient and may be used only by the recipient, and then only for (1) research use, (2) discovery, development and monitoring of biologic drugs and vaccines, (3) quality assurance testing of biologic drugs and vaccines, and (4) product release assays for biologic drugs and vaccines. No other commercial use is allowed. "Commercial use” means any and all uses of this product by recipient for monetary or other consideration, including providing a service, information or data to unaffiliated third parties, and resale of this product for any use. Recipient has no right to propagate, modify, derivatize, genetically engineer or otherwise create variations of the cells or genes stably transfected within the cells. In addition, recipient must use Bio-Glo™ Luciferase Assay System purchased from Promega Corporation for all luminescence assays using this product or contact Promega to obtain a license for use of this product with reagents other than Promega’s. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING AS TO MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.

U.S. Pat. No. 10,077,244 and other patents.

Patent Pending.

U.S. Pat. No. 8,008,006 and European Pat. No. 1341808.

Product cannot be used for proficiency testing.

Licensed from Lonza Cologne GmbH under U.S. Pat. Nos. 7,700,357, 8,192,990 and 8,003,389, European Pat. Nos. 1297119, 1522587, 1607484 and 1741778 and other pending and issued patents.

Specifications

You are viewing: JA6705 Change Configuration

What's in the box?

Item Part # Size

TCR/CD3 Effector Cells (IL-2)

J129A 5 × 0.8ml

Fetal Bovine Serum

J121A 5 × 4ml

RPMI 1640 Medium

G708A 5 × 36ml

Bio-Glo™ Luciferase Assay Buffer

G719A 5 × 10ml

Bio-Glo™ Luciferase Assay Substrate

G720A 5 × 1 vial

SDS

Choose language:

Download SDSPDF (314 KB) – English (United States)

Certificate of Analysis

Search by lot number

Use Restrictions

Not For Medical Diagnostic Use.

Storage Conditions

Please see the Protocol for detailed storage conditions.

Patents and Disclaimers

NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE STATEMENT. If the recipient is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the recipient with a full refund.

This product may not be further sold or transferred by the recipient and may be used only by the recipient, and then only for (1) research use, (2) discovery, development and monitoring of biologic drugs and vaccines, (3) quality assurance testing of biologic drugs and vaccines, and (4) product release assays for biologic drugs and vaccines. No other commercial use is allowed. "Commercial use” means any and all uses of this product by recipient for monetary or other consideration, including providing a service, information or data to unaffiliated third parties, and resale of this product for any use. Recipient has no right to propagate, modify, derivatize, genetically engineer or otherwise create variations of the cells or genes stably transfected within the cells. In addition, recipient must use Bio-Glo™ Luciferase Assay System purchased from Promega Corporation for all luminescence assays using this product or contact Promega to obtain a license for use of this product with reagents other than Promega’s. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING AS TO MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.

U.S. Pat. No. 10,077,244 and other patents.

Patent Pending.

U.S. Pat. No. 8,008,006 and European Pat. No. 1341808.

Product cannot be used for proficiency testing.

Licensed from Lonza Cologne GmbH under U.S. Pat. Nos. 7,700,357, 8,192,990 and 8,003,389, European Pat. Nos. 1297119, 1522587, 1607484 and 1741778 and other pending and issued patents.

Specifications

You are viewing: JA1072 Change Configuration

What's in the box?

Item Part # Size

TCR/CD3 Effector Cells (CPM) (IL-2)

J127A 2 × 1ml

FcγRIIb aAPC/CHO-K1 (CPM)

J325A 2 × 1ml

SDS

Choose language:

Download SDSPDF (298 KB) – English (United States)

Certificate of Analysis

Search by lot number

Use Restrictions

Not For Medical Diagnostic Use.

Storage Conditions

Please see the Protocol for detailed storage conditions.

Patents and Disclaimers

NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE STATEMENT. If the recipient is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the recipient with a full refund.

This product may not be further sold or transferred by the recipient and may be used only by the recipient, and then only for (1) research use, (2) discovery, development and monitoring of biologic drugs and vaccines, (3) quality assurance testing of biologic drugs and vaccines, and (4) product release assays for biologic drugs and vaccines. No other commercial use is allowed. "Commercial use" means any and all uses of this product by recipient for monetary or other consideration, including providing a service, information or data to unaffiliated third parties, and resale of this product for any use. Recipient has no right to modify, derivatize, genetically engineer or otherwise create variations of the cells or genes stably transfected within the cells except that recipient may propagate and store the cells for long-term use. In addition, recipient must use Bio-Glo™ Luciferase Assay System purchased from Promega Corporation for all luminescence assays using this product or contact Promega to obtain a license for use of this product with reagents other than Promega's. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING AS TO MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.

U.S. Pat. No. 8,008,006 and European Pat. No. 1341808.

Licensed from Lonza Cologne GmbH under U.S. Pat. Nos. 7,700,357, 8,192,990 and 8,003,389, European Pat. Nos. 1297119, 1522587, 1607484 and 1741778 and other pending and issued patents.

Product cannot be used for proficiency testing.

Specifications

You are viewing: K1231 Change Configuration

What's in the box?

Item Part # Size

Control Ab, Anti-CD28

K123A 1 × 1 each

SDS

Choose language:

Download SDSPDF (188 KB) – English (United States)

Certificate of Analysis

Search by lot number

Use Restrictions

Not For Medical Diagnostic Use.

Storage Conditions

BB

Please see the Protocol for detailed storage conditions.

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